WEBVTT 00:00:00.500 --> 00:00:02.640 The following content is provided under a Creative 00:00:02.640 --> 00:00:04.210 Commons license. 00:00:04.210 --> 00:00:06.510 Your support will help MIT Open Courseware 00:00:06.510 --> 00:00:10.840 continue to offer high quality educational resources for free. 00:00:10.840 --> 00:00:13.500 To make a donation or view additional materials 00:00:13.500 --> 00:00:17.440 from hundreds of MIT courses, visit MIT Open Courseware 00:00:17.440 --> 00:00:18.410 at ocw.mit.edu. 00:00:25.173 --> 00:00:26.840 ELIZABETH NOLAN: We're going to continue 00:00:26.840 --> 00:00:29.690 where we left off last time. 00:00:29.690 --> 00:00:33.470 So briefly I'll make a few points about initiation 00:00:33.470 --> 00:00:35.487 of translation in prokaryotes. 00:00:35.487 --> 00:00:37.820 And then where we're going to spend the bulk of the time 00:00:37.820 --> 00:00:42.380 today is with a review of tRNAs and then discussing 00:00:42.380 --> 00:00:44.980 the aminoacyl-tRNA synthetases, which 00:00:44.980 --> 00:00:48.020 are the enzymes responsible for loading 00:00:48.020 --> 00:00:51.600 amino acids onto the three prime end of the tRNA. 00:00:51.600 --> 00:00:55.100 And these points are important because these process has 00:00:55.100 --> 00:00:57.350 to happen in order for the amino acids 00:00:57.350 --> 00:00:59.750 to be delivered to the ribosome, which 00:00:59.750 --> 00:01:02.430 is where we'll go on Wednesday. 00:01:02.430 --> 00:01:07.970 So the first questions are, how does initiation happen? 00:01:07.970 --> 00:01:12.020 So how does this ribosome, 70S ribosome, 00:01:12.020 --> 00:01:16.280 get assembled with the mRNA and initiator tRNA bound? 00:01:16.280 --> 00:01:17.990 And then we're going to ask, how do we 00:01:17.990 --> 00:01:21.980 get an aminoacyl-tRNA, such that the amino acids can 00:01:21.980 --> 00:01:24.770 be delivered to the ribosome? 00:01:24.770 --> 00:01:29.600 So first, for initiation in prokaryotes, 00:01:29.600 --> 00:01:31.640 there's a few steps to this process. 00:01:31.640 --> 00:01:34.700 We'll just look at these at a basically superficial level 00:01:34.700 --> 00:01:35.630 of detail. 00:01:35.630 --> 00:01:39.110 But recall that there are translation factors. 00:01:39.110 --> 00:01:42.610 And during initiation, there are three initiation factors-- 00:01:42.610 --> 00:01:45.020 so IF 1, 2, and 3-- 00:01:45.020 --> 00:01:50.000 that are required to help assemble the 70S ribosome here. 00:01:50.000 --> 00:01:54.800 So first in terms of initiation, what happens 00:01:54.800 --> 00:02:01.650 is that the mRNA needs to bind to the 16S RNA of the 30S 00:02:01.650 --> 00:02:02.150 subunit. 00:02:10.100 --> 00:02:21.560 And so I point this out because at this stage in the process, 00:02:21.560 --> 00:02:24.350 the 70S ribosome isn't assembled yet. 00:02:24.350 --> 00:02:29.600 So we have the mRNA binding to the small subunit. 00:02:29.600 --> 00:02:37.820 And this process requires initiation factor 3. 00:02:37.820 --> 00:02:41.600 And effectively what happens is that the mRNA has 00:02:41.600 --> 00:02:44.000 a region called the Shine-Dalgarno sequence 00:02:44.000 --> 00:02:47.300 in prokaryotes, which is the site of ribosome binding. 00:02:47.300 --> 00:02:49.490 And then upstream of that is a start codon 00:02:49.490 --> 00:02:52.080 that signals for the start of translation. 00:02:52.080 --> 00:02:58.290 So if we think about the mRNA of the five prime end, 00:02:58.290 --> 00:03:06.550 and somewhere there's a sequence that 00:03:06.550 --> 00:03:07.990 signals for ribosome binding. 00:03:15.540 --> 00:03:21.790 OK, and then we have our start codon 00:03:21.790 --> 00:03:23.860 that signals the start of translation. 00:03:27.890 --> 00:03:30.360 OK. 00:03:30.360 --> 00:03:36.620 And so this gets translated here. 00:03:36.620 --> 00:03:40.640 OK, so this start codon pairs with initiator tRNA. 00:03:47.360 --> 00:03:50.090 And this initiator tRNA is special. 00:03:50.090 --> 00:03:53.930 One reason why it's special is because the amino acid attached 00:03:53.930 --> 00:03:56.825 is an N-Formylmethionine OK. 00:04:13.420 --> 00:04:20.079 So sometimes the initiator tRNA is called f-met tRNA f-met 00:04:20.079 --> 00:04:23.650 as an abbreviation there. 00:04:23.650 --> 00:04:27.610 So just as some overview here, what 00:04:27.610 --> 00:04:29.440 we're seeing in this alignment is 00:04:29.440 --> 00:04:31.750 a number of the ribosome binding sites, 00:04:31.750 --> 00:04:35.230 or Shine-Dalgarno sequences in prokaryotes. 00:04:35.230 --> 00:04:39.400 We have the start codon on that pairs with the initiator tRNA. 00:04:39.400 --> 00:04:43.330 And here's a schematic depiction of what I've indicated here 00:04:43.330 --> 00:04:44.800 on the board. 00:04:44.800 --> 00:04:50.020 OK, so the mRNA binds to the 16S of the 30S subunit. 00:04:50.020 --> 00:04:53.230 So the 70S is not assembled at this stage. 00:04:53.230 --> 00:04:56.360 And IF3 is involved, as I said. 00:04:56.360 --> 00:05:00.550 The Shine-Dalgarno sequence determines the start site. 00:05:00.550 --> 00:05:03.330 And we determine the reading frame, as well. 00:05:03.330 --> 00:05:07.210 So here is just an indicating translation of a polypeptide. 00:05:07.210 --> 00:05:10.660 What happens after that? 00:05:10.660 --> 00:05:16.360 So after that, it's necessary to assemble the 70S ribosome, 00:05:16.360 --> 00:05:19.510 have the initiator tRNA in the P site, 00:05:19.510 --> 00:05:22.630 and have the cell ready to go for translation. 00:05:22.630 --> 00:05:25.450 And here's just one cartoon overview 00:05:25.450 --> 00:05:28.600 that we'll use as a description of this process. 00:05:28.600 --> 00:05:29.710 OK. 00:05:29.710 --> 00:05:31.210 So what do we see? 00:05:31.210 --> 00:05:33.520 We've talked about this step so far. 00:05:33.520 --> 00:05:37.190 We see there's a role for initiation factor 1. 00:05:37.190 --> 00:05:40.570 And in this cartoon, if we imagine the E site, 00:05:40.570 --> 00:05:44.050 the P site, and the A site, what we see 00:05:44.050 --> 00:05:48.160 is that IF1 is binding to the site of the ribosome. 00:05:48.160 --> 00:05:49.930 And one way we can think about this 00:05:49.930 --> 00:05:53.930 is that the initiator tRNA has to get to the P site. 00:05:53.930 --> 00:05:57.460 And so that region is blocked to facilitate the initiator 00:05:57.460 --> 00:05:59.940 tRNA getting to the P site. 00:05:59.940 --> 00:06:04.630 OK, we see that initiator tRNA binding to the P site. 00:06:04.630 --> 00:06:07.600 And this happens via formation of a ternary complex 00:06:07.600 --> 00:06:10.600 with IF2 and GTP. 00:06:10.600 --> 00:06:15.640 So initiation factor 2 hydrolyzes GTP. 00:06:15.640 --> 00:06:18.910 There's an event that results in joining of the two subunits. 00:06:18.910 --> 00:06:21.250 And there has to be dissociation of these initiation 00:06:21.250 --> 00:06:24.550 factors for the ribosome to be ready to accept 00:06:24.550 --> 00:06:28.090 its first aminoacyl-tRNA in the A site. 00:06:28.090 --> 00:06:31.120 OK, so the outcome of this process 00:06:31.120 --> 00:06:33.370 here is that we have an assembled 70S 00:06:33.370 --> 00:06:36.880 ribosome with the initiator tRNA in the P site. 00:06:36.880 --> 00:06:40.270 The A site is empty, so it can accommodate 00:06:40.270 --> 00:06:43.300 an incoming aminoacyl-tRNA. 00:06:43.300 --> 00:06:47.120 And the E site or exit site is also empty. 00:06:47.120 --> 00:06:49.840 So that's the main take home for initiation. 00:06:49.840 --> 00:06:51.340 And that's the extent to which we're 00:06:51.340 --> 00:06:54.770 going to discuss it within this class. 00:06:54.770 --> 00:06:57.520 So in order to get to the elongation cycle, 00:06:57.520 --> 00:07:01.200 we need to get the aminoacyl-tRNA into the A site. 00:07:01.200 --> 00:07:05.410 And that's going to require the help of EF-Tu, so elongation 00:07:05.410 --> 00:07:07.630 factor Tu. 00:07:07.630 --> 00:07:10.660 Before we discuss how elongation factor 00:07:10.660 --> 00:07:14.240 Tu is going to help deliver that aminoacyl-tRNA, 00:07:14.240 --> 00:07:16.810 we need to talk about how we get the aminoacyl-tRNA 00:07:16.810 --> 00:07:18.230 in the first place. 00:07:18.230 --> 00:07:21.610 So what is the tRNA structure, just as a review 00:07:21.610 --> 00:07:23.810 to get everyone up to speed. 00:07:23.810 --> 00:07:27.730 How are amino acid monomers attached to the tRNA? 00:07:27.730 --> 00:07:30.080 And how is the correct amino acid attached? 00:07:30.080 --> 00:07:31.780 So this is an aspect of fidelity, 00:07:31.780 --> 00:07:36.040 which came up as a concept last week in lecture. 00:07:36.040 --> 00:07:37.540 And so we'll look at the mechanism 00:07:37.540 --> 00:07:40.180 of aminoacyl-tRNA synthetases to see 00:07:40.180 --> 00:07:42.760 how is the correct amino acid attached, 00:07:42.760 --> 00:07:46.450 and then what happens if the wrong amino acid is selected. 00:07:46.450 --> 00:07:48.400 Are there mechanisms to correct that? 00:07:48.400 --> 00:07:52.430 And if it's not corrected, what are the consequences here? 00:07:52.430 --> 00:07:59.900 So moving forward with that, we're 00:07:59.900 --> 00:08:05.340 going to focus on the tRNAs and addressing those questions. 00:08:05.340 --> 00:08:09.260 So just as a review, so we can think 00:08:09.260 --> 00:08:18.990 about tRNA secondary structure, which is often 00:08:18.990 --> 00:08:20.430 described as cloverleaf. 00:08:32.049 --> 00:08:33.620 So we have a five prime end. 00:08:37.110 --> 00:08:38.970 The tRNA has several arms. 00:08:53.750 --> 00:08:54.470 OK. 00:08:54.470 --> 00:08:55.680 So we have a D arm. 00:08:58.430 --> 00:09:01.610 This arm here has the anticodon that pairs 00:09:01.610 --> 00:09:02.850 with the codon of the mRNA. 00:09:08.310 --> 00:09:18.550 We have a variable arm, this arm here. 00:09:18.550 --> 00:09:24.190 And we have this three prime end here, 00:09:24.190 --> 00:09:27.860 where the amino acids get attached. 00:09:27.860 --> 00:09:30.640 So this, in terms of base numbering, 00:09:30.640 --> 00:09:36.370 we have C74, C75, A76 here. 00:09:38.780 --> 00:09:39.280 OH. 00:09:39.280 --> 00:09:42.415 This is often called the CCA acceptor stem. 00:09:48.130 --> 00:09:50.470 And the amino acids are attached here. 00:09:50.470 --> 00:09:51.970 I'm going to abbreviate amino acid 00:09:51.970 --> 00:10:00.935 as AA via an ester linkage. 00:10:05.820 --> 00:10:08.470 And these ester linkages are important for the chemistry 00:10:08.470 --> 00:10:10.490 that happens in the ribosome. 00:10:10.490 --> 00:10:10.990 OK. 00:10:10.990 --> 00:10:16.570 So we can imagine just if we have abbreviating the tRNA 00:10:16.570 --> 00:10:24.110 structure like this and if we think about the sugar of A76-- 00:10:32.370 --> 00:10:32.910 bless you. 00:10:35.660 --> 00:10:37.140 OK. 00:10:37.140 --> 00:10:44.580 We have one prime, two prime, three prime here. 00:10:44.580 --> 00:10:47.370 This type of connectivity here. 00:10:47.370 --> 00:10:51.652 And this is abbreviated throughout as amino acid tRNA, 00:10:51.652 --> 00:10:56.730 aa in general terms, or the three-letter abbreviations, 00:10:56.730 --> 00:11:01.980 like what we saw for f-met tRNA f-met with the initiator tRNA 00:11:01.980 --> 00:11:02.480 here. 00:11:07.210 --> 00:11:10.860 So here's a schematic of a tRNA secondary structure with a bit 00:11:10.860 --> 00:11:13.140 more detail than what I show you on the board. 00:11:15.960 --> 00:11:17.880 And something we need to keep in mind 00:11:17.880 --> 00:11:22.020 is even though we often draw the tRNA in this cloverleaf type 00:11:22.020 --> 00:11:24.660 depiction, it has tertiary structure. 00:11:24.660 --> 00:11:27.660 And so it's very important to think about this structure 00:11:27.660 --> 00:11:31.830 as we think about how the tRNAs enter the various sites 00:11:31.830 --> 00:11:33.160 of the ribosome. 00:11:33.160 --> 00:11:33.660 OK. 00:11:33.660 --> 00:11:36.540 So this structure is L-shaped. 00:11:36.540 --> 00:11:39.780 And I like this depiction here because regions 00:11:39.780 --> 00:11:41.580 of the secondary structure are color 00:11:41.580 --> 00:11:44.280 coded with the corresponding regions 00:11:44.280 --> 00:11:46.820 of this tertiary structure here. 00:11:46.820 --> 00:11:49.380 OK, so we see the shell shape of an L, 00:11:49.380 --> 00:11:53.370 rather upside down here, where we have the CCA acceptor stem 00:11:53.370 --> 00:12:01.720 over here and the anticodon arm and anticodon region down here. 00:12:01.720 --> 00:12:06.900 So what is a consequence of this structure? 00:12:06.900 --> 00:12:09.690 The tRNA is quite narrow. 00:12:09.690 --> 00:12:15.070 So we're thinking about 20 to 25 Angstroms in width. 00:12:15.070 --> 00:12:18.540 And if we think about this in the context of the ribosome 00:12:18.540 --> 00:12:21.210 and the peptidyl transferase center, 00:12:21.210 --> 00:12:24.930 3 tRNAs need to fit into that catalytic center 00:12:24.930 --> 00:12:26.970 during the elongation cycle. 00:12:26.970 --> 00:12:30.060 So it makes sense that they're relatively narrow. 00:12:30.060 --> 00:12:33.660 This allows three to fit there. 00:12:33.660 --> 00:12:37.350 So as we think about the translation process 00:12:37.350 --> 00:12:40.110 and also think about some of the translation factors, 00:12:40.110 --> 00:12:43.320 we want to keep this type of structure in mind here. 00:12:47.820 --> 00:12:51.540 Here's just another view of that, 00:12:51.540 --> 00:12:53.610 with some additional descriptions 00:12:53.610 --> 00:12:55.530 of the overall structure. 00:12:55.530 --> 00:13:00.030 And this includes the numbering of the tRNA bases 00:13:00.030 --> 00:13:01.230 within that structure here. 00:13:03.840 --> 00:13:06.810 Just a point to make, this won't be a major focal point 00:13:06.810 --> 00:13:08.940 in the course, but do keep in mind 00:13:08.940 --> 00:13:12.480 that tRNA contains many post-transcriptionally modified 00:13:12.480 --> 00:13:16.560 bases, so you'll see an example of that in problem set one. 00:13:16.560 --> 00:13:19.890 Up to 25% of the bases can be modified. 00:13:19.890 --> 00:13:25.000 Typically, we see about 5% to 20% of them modified here. 00:13:25.000 --> 00:13:27.570 OK, you're not responsible for these structures, 00:13:27.570 --> 00:13:29.940 these modified structures, in the context of this class. 00:13:32.980 --> 00:13:36.460 So the key question for today is how 00:13:36.460 --> 00:13:42.870 are amino acids attached to the tRNA, as shown here? 00:13:42.870 --> 00:13:45.210 And in order for that to happen, there's 00:13:45.210 --> 00:13:50.240 a family of enzymes called aminoacyl-tRNA synthetases, 00:13:50.240 --> 00:13:53.330 or abbreviated aaRS. 00:13:53.330 --> 00:13:56.370 OK, so this name tells you right away, synthetase, 00:13:56.370 --> 00:14:00.090 that these enzymes use ATP. 00:14:00.090 --> 00:14:03.270 And these enzymes catalyze the attachment 00:14:03.270 --> 00:14:06.630 of amino acids to the three prime OH, 00:14:06.630 --> 00:14:13.230 or sometimes two prime OH, of the tRNA here for that. 00:14:13.230 --> 00:14:16.630 And so we're going to consider this overall reaction. 00:14:16.630 --> 00:14:18.630 And then we're going to think about the reaction 00:14:18.630 --> 00:14:22.950 mechanism and experiments that were done to give support 00:14:22.950 --> 00:14:27.090 to the mechanism that we see. 00:14:27.090 --> 00:14:30.030 So all aminoacyl-tRNA synthetases 00:14:30.030 --> 00:14:37.410 require ATP and hydrolyze ATP to AMP and PPI. 00:14:37.410 --> 00:14:40.140 And so they catalyze this overall reaction 00:14:40.140 --> 00:14:42.630 where we have an amino acid monomer. 00:14:42.630 --> 00:14:46.470 We have the tRNA that encodes this-- 00:14:46.470 --> 00:14:48.870 that is for this amino acid. 00:14:48.870 --> 00:14:56.220 ATP to give us the aminoacyl-tRNA AMP and PPI. 00:14:56.220 --> 00:15:00.740 So if the ATP is being hydrolyzed to AMP and PPI, 00:15:00.740 --> 00:15:02.730 what phosphate is being attacked? 00:15:02.730 --> 00:15:05.710 So we saw on Friday there's the alpha, beta, 00:15:05.710 --> 00:15:07.050 and gamma phosphates of ATP. 00:15:23.480 --> 00:15:25.740 OK, pardon? 00:15:25.740 --> 00:15:26.420 AUDIENCE: Beta. 00:15:26.420 --> 00:15:27.770 ELIZABETH NOLAN: Beta. 00:15:27.770 --> 00:15:29.042 Any takers? 00:15:29.042 --> 00:15:30.770 AUDIENCE: Alpha? 00:15:30.770 --> 00:15:32.820 ELIZABETH NOLAN: Any takers? 00:15:32.820 --> 00:15:33.320 Gamma? 00:15:36.790 --> 00:15:38.550 Yeah, so it's alpha. 00:15:38.550 --> 00:15:43.590 If you're getting AMP, it's attack at alpha. 00:15:43.590 --> 00:15:48.360 If you're getting ADP, it's attack at gamma here. 00:15:57.260 --> 00:16:01.860 OK, so P alpha is next door to the ribose of the nuc-- 00:16:01.860 --> 00:16:02.590 there. 00:16:02.590 --> 00:16:03.880 Yeah. 00:16:03.880 --> 00:16:06.470 OK. 00:16:06.470 --> 00:16:15.230 So if we consider this overall reaction, how does it work? 00:16:15.230 --> 00:16:18.710 Just before that, one other observation 00:16:18.710 --> 00:16:21.050 I just want to point out, if we're 00:16:21.050 --> 00:16:25.190 thinking about these enzymes and asking 00:16:25.190 --> 00:16:28.250 what is it that they recognize of the tRNA, 00:16:28.250 --> 00:16:30.140 so we have the anticodon. 00:16:30.140 --> 00:16:33.020 And that goes in hand-in-hand with the identity 00:16:33.020 --> 00:16:34.640 of the amino acid. 00:16:34.640 --> 00:16:37.370 Just keep in mind that it's not just the anticodon. 00:16:37.370 --> 00:16:41.930 So here we're seeing an example of an aminoacyl-tRNA synthetase 00:16:41.930 --> 00:16:44.240 with its tRNA bound. 00:16:44.240 --> 00:16:47.420 And we see that there's many contacts between the tRNA 00:16:47.420 --> 00:16:49.250 and this enzyme here. 00:16:49.250 --> 00:16:52.730 OK, so here we have the amino acid end, the anti-codon end, 00:16:52.730 --> 00:16:53.750 and all throughout here. 00:16:59.460 --> 00:17:05.220 So what is the mechanism to get us where we need to go? 00:17:05.220 --> 00:17:09.170 We have our overall reaction that I'll put up on the board, 00:17:09.170 --> 00:17:12.270 just to keep it straight as we move forward. 00:17:12.270 --> 00:17:25.529 So amino acid plus ATP plus the tRNA for that amino acid. 00:17:29.840 --> 00:17:40.430 Aminoacyl-tRNA synthetase to give us the aminoacyl-tRNA 00:17:40.430 --> 00:17:44.495 plus AMP plus PPI. 00:17:47.280 --> 00:17:48.990 So let's consider a mechanism. 00:17:55.350 --> 00:17:57.510 This is going to be a two-step mechanism. 00:18:00.800 --> 00:18:03.030 And so in the first step of this mechanism, 00:18:03.030 --> 00:18:20.180 we have the amino acid plus ATP. 00:18:24.940 --> 00:18:31.585 And we have formation of an OAMP intermediate. 00:18:37.760 --> 00:18:40.940 Plus PPI here. 00:18:40.940 --> 00:18:50.091 So this intermediate is called an amino adenylate. 00:18:55.650 --> 00:18:57.480 Adenlyate because adenosine here. 00:19:00.820 --> 00:19:04.260 And we need to think about why this intermediate might form. 00:19:04.260 --> 00:19:07.200 Why would we propose this in a mechanism? 00:19:07.200 --> 00:19:09.850 And then in step two-- 00:19:09.850 --> 00:19:12.540 we'll come back to that in a minute-- 00:19:12.540 --> 00:19:31.050 we can take our amino adenlyate, have our tRNA, 00:19:31.050 --> 00:19:35.700 this is the three prime end here. 00:19:35.700 --> 00:19:55.460 We can have attack with release of AMP. 00:19:55.460 --> 00:20:04.390 OK, so here we have the ester linkage at the three prime end, 00:20:04.390 --> 00:20:06.960 like what we see on that board here, 00:20:06.960 --> 00:20:11.060 to give us our aminoacyl-tRNA. 00:20:11.060 --> 00:20:13.330 OK, so we see in step one, there's 00:20:13.330 --> 00:20:16.840 formation of this amino adenylade intermediate. 00:20:16.840 --> 00:20:19.330 And in step two, there's transfer 00:20:19.330 --> 00:20:23.230 of the amino acid monomer to the three prime end of the tRNA 00:20:23.230 --> 00:20:25.330 here. 00:20:25.330 --> 00:20:32.780 So why might these enzymes go through that OAMP intermediate? 00:20:32.780 --> 00:20:35.190 What needs to happen for this chemistry to occur? 00:20:45.208 --> 00:20:47.250 AUDIENCE: You need a more activated reading group 00:20:47.250 --> 00:20:50.040 to have that acyl substitution form an ester 00:20:50.040 --> 00:20:51.292 from a carboxylate. 00:20:51.292 --> 00:20:52.250 ELIZABETH NOLAN: Right. 00:20:52.250 --> 00:20:56.100 We need to activate the CO2H group there. 00:20:56.100 --> 00:20:58.830 So this affords that. 00:20:58.830 --> 00:21:02.140 So what might be another possible mechanism, right? 00:21:02.140 --> 00:21:03.930 Imagine you're the experimentalist 00:21:03.930 --> 00:21:07.860 and you've combined your eighth amino acid ATP 00:21:07.860 --> 00:21:12.990 tRNA and this enzyme you've isolated in a test tube. 00:21:12.990 --> 00:21:16.270 And you see you've got this as a product. 00:21:16.270 --> 00:21:18.000 And this as a product. 00:21:18.000 --> 00:21:21.660 And you're wondering how did we get from reactants to products? 00:21:21.660 --> 00:21:23.460 This is one possibility. 00:21:23.460 --> 00:21:26.790 Maybe there's also a possibility of a concerted mechanism 00:21:26.790 --> 00:21:29.760 where there's no intermediate like the one I'm showing you 00:21:29.760 --> 00:21:30.660 here. 00:21:30.660 --> 00:21:32.220 These are just things to keep in mind 00:21:32.220 --> 00:21:34.680 when thinking about reactions. 00:21:34.680 --> 00:21:37.770 This two-step mechanism is the accepted mechanism 00:21:37.770 --> 00:21:40.440 for the amino aceyl tRNA synthetases. 00:21:40.440 --> 00:21:42.090 And so what we're going to think about 00:21:42.090 --> 00:21:45.120 are what are the experiments that were done 00:21:45.120 --> 00:21:49.170 to support this mechanism here. 00:21:49.170 --> 00:21:51.210 So what are the things we need to think about? 00:21:51.210 --> 00:21:52.793 And so we're going to think about this 00:21:52.793 --> 00:21:57.900 by examining one aminoacyl-tRNA synthetase as a paradigm. 00:21:57.900 --> 00:22:01.368 And this is the one for a isoleucine here. 00:22:28.180 --> 00:22:30.380 OK, so what are the experiments that 00:22:30.380 --> 00:22:33.290 need to be done to characterize this reaction 00:22:33.290 --> 00:22:34.640 and determine mechanism? 00:22:58.100 --> 00:22:59.190 OK. 00:22:59.190 --> 00:23:04.800 So one thing we need to confirm is reaction stoichiometry. 00:23:04.800 --> 00:23:08.460 So there's a stoichiometry up in what I've written above. 00:23:08.460 --> 00:23:12.250 But experimentally, that needs to be determined. 00:23:12.250 --> 00:23:21.360 So one, reaction stoichiometry. 00:23:21.360 --> 00:23:23.720 And so how can we think about this? 00:23:23.720 --> 00:23:27.020 We can think about the equivalence of the amino acid. 00:23:27.020 --> 00:23:28.900 So in this case, isoleucine. 00:23:28.900 --> 00:23:30.800 How many equivalents of isoleucine? 00:23:44.330 --> 00:23:47.410 And presumably, this isoleucine binds to the enzymes. 00:23:47.410 --> 00:23:48.910 We can think about it of equivalence 00:23:48.910 --> 00:23:50.620 of isoleucine bound. 00:23:50.620 --> 00:23:53.380 And we also see that ATP is consumed, right? 00:23:53.380 --> 00:23:55.690 That's hydrolyzed to AMP and PPI. 00:23:55.690 --> 00:24:00.100 So how many equivalents of ATP are consumed in this reaction? 00:24:13.990 --> 00:24:16.000 What else do we want to know? 00:24:16.000 --> 00:24:18.880 We need to know something about kinetics. 00:24:18.880 --> 00:24:21.850 So what are rates of formation? 00:24:21.850 --> 00:24:24.820 What is the rate of formation of the product, 00:24:24.820 --> 00:24:28.120 the aminoacyl-tRNA, and since I've 00:24:28.120 --> 00:24:30.250 told you this intermediate forms, 00:24:30.250 --> 00:24:33.736 what is the rate of formation of the intermediate? 00:24:33.736 --> 00:24:35.440 And since this is an intermediate, 00:24:35.440 --> 00:24:36.690 it's something transient. 00:24:36.690 --> 00:24:40.060 So we need to think about how are we as experimentalists 00:24:40.060 --> 00:24:42.370 going to detect this intermediate over the course 00:24:42.370 --> 00:24:44.590 of this reaction. 00:24:44.590 --> 00:24:48.280 It forms and decays in order to get product here. 00:24:52.850 --> 00:24:54.320 So rates of formation. 00:25:00.590 --> 00:25:14.335 And so we have formation of our product, which in this case-- 00:25:21.580 --> 00:25:35.292 and then formation of the intermediate, which 00:25:35.292 --> 00:25:36.500 I'll just abbreviate Ile-AMP. 00:25:40.930 --> 00:25:42.590 And what else would we like to know? 00:25:45.290 --> 00:25:47.720 We can figure out how, in addition 00:25:47.720 --> 00:25:52.490 to rate of formation of the product and the intermediate, 00:25:52.490 --> 00:26:06.430 we can think about the rate of transfer of Ile 00:26:06.430 --> 00:26:09.025 from the intermediate to the tRNA. 00:26:13.000 --> 00:26:16.330 So what this tells us is that we need 00:26:16.330 --> 00:26:26.965 a way to look for or detect the intermediate. 00:26:33.740 --> 00:26:34.240 Here. 00:26:39.100 --> 00:26:43.930 So imagine let's just have a hypothetical situation. 00:26:43.930 --> 00:26:47.110 If we find the intermediate, that tells us 00:26:47.110 --> 00:26:49.330 something about the reaction. 00:26:49.330 --> 00:26:51.005 If we don't find the intermediate, 00:26:51.005 --> 00:26:51.880 what can we conclude? 00:26:55.998 --> 00:26:56.960 Pardon? 00:26:56.960 --> 00:27:00.288 AUDIENCE: That there was no intermediate? 00:27:00.288 --> 00:27:02.080 ELIZABETH NOLAN: So that's one possibility. 00:27:02.080 --> 00:27:04.570 Are there other possibilities if our method doesn't 00:27:04.570 --> 00:27:07.840 let us detect the intermediate? 00:27:07.840 --> 00:27:09.989 AUDIENCE: Second step is to test. 00:27:13.250 --> 00:27:16.790 ELIZABETH NOLAN: Can it be hard to detect an intermediate? 00:27:16.790 --> 00:27:19.250 It can be very hard, right? 00:27:19.250 --> 00:27:21.830 So they don't always-- 00:27:21.830 --> 00:27:23.690 there aren't around all the time very much 00:27:23.690 --> 00:27:26.240 or in very abundant quantities. 00:27:26.240 --> 00:27:30.650 So if it's not detected, could it be there? 00:27:30.650 --> 00:27:31.850 Yeah, it might be there. 00:27:31.850 --> 00:27:35.730 And the method just didn't allow for it to be seen. 00:27:35.730 --> 00:27:38.743 So you always need to keep that possibility in mind. 00:27:38.743 --> 00:27:40.160 This will be a case where there is 00:27:40.160 --> 00:27:42.800 a robust method that allows us to detect 00:27:42.800 --> 00:27:44.980 this type of intermediate. 00:27:44.980 --> 00:27:47.420 But always keep that in mind. 00:27:47.420 --> 00:27:52.880 OK, so first thinking about reaction stoichiometry. 00:27:52.880 --> 00:27:55.340 We're not going to go over the experiments that 00:27:55.340 --> 00:27:56.930 were done to define this. 00:27:56.930 --> 00:27:59.450 I'll just tell you some facts that result 00:27:59.450 --> 00:28:01.890 from some experimental studies. 00:28:01.890 --> 00:28:06.740 So this isoleucine aminoacyl-tRNA synthetase 00:28:06.740 --> 00:28:09.560 binds 1 equivalent of isoleucine as indicated 00:28:09.560 --> 00:28:11.750 in the overall reaction. 00:28:11.750 --> 00:28:15.380 And it consumes one equivalent of ATP, 00:28:15.380 --> 00:28:17.870 also as shown in this overall reaction, 00:28:17.870 --> 00:28:21.860 to make one equivalent of the aminoacyl-tRNA. 00:28:21.860 --> 00:28:26.760 OK and these stoichiometries were determined experimentally. 00:28:26.760 --> 00:28:31.010 So now we need to think about points two and three 00:28:31.010 --> 00:28:33.840 to characterize the reaction kinetics. 00:28:33.840 --> 00:28:36.242 So what experiments were done? 00:28:36.242 --> 00:28:37.700 So there are several different sets 00:28:37.700 --> 00:28:40.040 of experiments, some of which we're familiar 00:28:40.040 --> 00:28:44.120 with from 7.05 or 5.07 and others that will be new 00:28:44.120 --> 00:28:46.700 and presented in more detail in recitation this week 00:28:46.700 --> 00:28:48.090 and next week. 00:28:48.090 --> 00:28:52.580 So we can imagine doing steady state kinetic experiments, 00:28:52.580 --> 00:28:55.880 as well as pre-steady state kinetic experiments. 00:28:55.880 --> 00:28:58.280 And the general aims here are, one, 00:28:58.280 --> 00:29:02.450 to determine the rate of aminoacyl-tRNA formation, 00:29:02.450 --> 00:29:05.480 to determine the rate of amino adenylate formation, 00:29:05.480 --> 00:29:07.160 so this intermediate-- and again, we 00:29:07.160 --> 00:29:09.800 need a method to detect the intermediate. 00:29:09.800 --> 00:29:11.300 And at the end of the day, we'd like 00:29:11.300 --> 00:29:15.380 to know what is the rate determining step. 00:29:15.380 --> 00:29:18.470 So a method that is commonly employed 00:29:18.470 --> 00:29:20.360 for these types of studies involves 00:29:20.360 --> 00:29:22.460 the use of radioactivity. 00:29:22.460 --> 00:29:27.020 And we'll just go over a few points about radioactivity now 00:29:27.020 --> 00:29:28.940 to help with understanding these experiments. 00:29:28.940 --> 00:29:33.110 And you'll hear more about this method in recitation this week. 00:29:33.110 --> 00:29:35.750 So the experiments I'm going to tell you about 00:29:35.750 --> 00:29:42.800 are going to involve the use of radio isotopes like C14, P32. 00:29:42.800 --> 00:29:45.140 And the question is, why do we like 00:29:45.140 --> 00:29:48.980 to use radio isotopes in biochemical experiments? 00:29:48.980 --> 00:29:51.170 And they're really excellent probes. 00:29:51.170 --> 00:29:53.000 It's the bottom line. 00:29:53.000 --> 00:29:56.420 And one reason for that is that if you can use a radio isotope 00:29:56.420 --> 00:30:00.200 like C14 or P32, it's introducing 00:30:00.200 --> 00:30:04.250 minimal perturbation into your system. 00:30:04.250 --> 00:30:07.490 So you're not needing to attach a fluorophore whether it be 00:30:07.490 --> 00:30:09.410 a small molecule or a protein. 00:30:09.410 --> 00:30:12.050 You're not modifying the structure 00:30:12.050 --> 00:30:14.910 of a component of your system. 00:30:14.910 --> 00:30:17.600 So the overall size and the chemical properties 00:30:17.600 --> 00:30:20.540 are maintained when you use different isotopes 00:30:20.540 --> 00:30:22.150 of the same element. 00:30:22.150 --> 00:30:24.510 And some of the ones we'll see today 00:30:24.510 --> 00:30:30.200 are, for instance, C14 labeled isoleucine, P32 labeled ATP. 00:30:30.200 --> 00:30:31.940 They have the same chemical properties 00:30:31.940 --> 00:30:34.850 as the unlabeled forms, and same size. 00:30:34.850 --> 00:30:36.800 The other point to make is that we 00:30:36.800 --> 00:30:40.590 can detect very small amounts of radioactivity in a sample. 00:30:40.590 --> 00:30:43.520 And you'll see some of those calculations 00:30:43.520 --> 00:30:46.490 and how to do them in recitation this week. 00:30:46.490 --> 00:30:49.010 So we can detect small amounts, and that's 00:30:49.010 --> 00:30:51.860 good for looking for something like an intermediate. 00:30:51.860 --> 00:30:55.910 And there's readily available techniques 00:30:55.910 --> 00:31:00.260 for quantifying radioactivity in a sample. 00:31:00.260 --> 00:31:02.450 So if you see nomenclature like this, 00:31:02.450 --> 00:31:06.110 the NX nomenclature indicates the radioisotope 00:31:06.110 --> 00:31:08.390 in this sample. 00:31:08.390 --> 00:31:11.860 And I'll just say in passing here, 00:31:11.860 --> 00:31:13.910 we all know the isotopes are atoms 00:31:13.910 --> 00:31:16.760 bearing the same number of protons but different numbers 00:31:16.760 --> 00:31:18.710 of neutrons. 00:31:18.710 --> 00:31:21.530 And radioactive isotopes have an unstable nucleus, 00:31:21.530 --> 00:31:24.740 which means there's a radioactive decay. 00:31:24.740 --> 00:31:29.140 And typically-- well, we often use beta emitters 00:31:29.140 --> 00:31:30.470 in biochemical studies. 00:31:30.470 --> 00:31:33.380 And that's what you'll see today. 00:31:33.380 --> 00:31:36.190 So what are some of the experiments? 00:31:38.720 --> 00:31:42.170 We're first going to consider looking at the steady state 00:31:42.170 --> 00:31:48.140 kinetics to ask what do we learn in the steady state. 00:31:56.970 --> 00:32:06.780 So from our steady state experiments, 00:32:06.780 --> 00:32:11.580 we're able to get our Kcat and our Km 00:32:11.580 --> 00:32:15.630 and the catalytic efficiency, which is the Kcat over Km. 00:32:15.630 --> 00:32:21.630 We're going to compare our Kcat values or turnover today. 00:32:21.630 --> 00:32:28.875 So experiment one is to monitor formation of product. 00:32:39.890 --> 00:32:41.690 So how is this done? 00:32:41.690 --> 00:32:45.110 This reaction is done by taking C14 labeled 00:32:45.110 --> 00:32:49.640 isoleucine and unlabeled tRNA and watching 00:32:49.640 --> 00:32:52.550 for transfer of that radio label to the tRNA. 00:33:14.550 --> 00:33:18.290 And so what comes from these studies 00:33:18.290 --> 00:33:23.410 is a Kcat on the order of 1.4 per second. 00:33:30.750 --> 00:33:35.970 And now we have a way to detect this amino adenylate 00:33:35.970 --> 00:33:36.930 intermediate. 00:33:36.930 --> 00:33:38.970 And we'll talk about that assay in a minute, 00:33:38.970 --> 00:33:41.370 after we get through this comparison. 00:33:41.370 --> 00:33:45.630 We do a steady state experiment to monitor 00:33:45.630 --> 00:33:52.652 formation of this amino adenylate intermediate. 00:33:55.200 --> 00:33:58.650 And this assay also uses radioactivity. 00:33:58.650 --> 00:34:04.425 And it's called ATP PPI exchange assay. 00:34:04.425 --> 00:34:06.300 And we'll go over how this works in a minute. 00:34:09.130 --> 00:34:11.760 So the results of these experiments 00:34:11.760 --> 00:34:17.909 give a Kcat on the order of 80 per second. 00:34:23.100 --> 00:34:25.790 So what does this comparison tell you? 00:34:36.550 --> 00:34:40.300 These values are quite different, correct? 00:34:40.300 --> 00:34:47.949 So we're seeing that this ATP PPI exchange assay 00:34:47.949 --> 00:34:51.489 is telling us that ATP PPI exchange, which 00:34:51.489 --> 00:34:54.130 is a measure of formation of this intermediate, 00:34:54.130 --> 00:35:00.010 is about 60-fold faster than formation of product here. 00:35:00.010 --> 00:35:03.910 That's an important observation to have. 00:35:03.910 --> 00:35:07.090 So how are we going to figure this out? 00:35:07.090 --> 00:35:10.180 How are we going to see this intermediate? 00:35:10.180 --> 00:35:13.750 That's the question we need to ask next. 00:35:13.750 --> 00:35:22.960 And so we need to go over this ATP PPI exchange assay. 00:35:22.960 --> 00:35:29.140 And this is an assay that will come up again in module 4 00:35:29.140 --> 00:35:32.230 when we talk about the biosynthesis 00:35:32.230 --> 00:35:33.770 of non-ribosomal peptides. 00:35:33.770 --> 00:35:36.835 So we'll return to this type of assay and data many times. 00:35:40.210 --> 00:36:02.080 So the question is, if we have this reaction, OK, 00:36:02.080 --> 00:36:03.400 how do we detect this? 00:36:08.590 --> 00:36:10.200 OK, it's not so easy. 00:36:10.200 --> 00:36:12.170 And we need an assay. 00:36:12.170 --> 00:36:14.150 And this is some of the background 00:36:14.150 --> 00:36:17.600 towards the development of this assay. 00:36:17.600 --> 00:36:23.720 So we need to suppose that our amino acid and ATP react 00:36:23.720 --> 00:36:26.330 with the aminoacyl-tRNA synthetase 00:36:26.330 --> 00:36:29.110 in the absence of tRNA. 00:36:29.110 --> 00:36:32.660 And that's indicated by step one, more or less. 00:36:32.660 --> 00:36:35.280 But that doesn't show it experimentally. 00:36:35.280 --> 00:36:38.540 So in the absence of tRNA, this amino acid and ATP 00:36:38.540 --> 00:36:42.770 react with the enzyme and they form the aminoacyl AMP 00:36:42.770 --> 00:36:45.260 intermediate and PPI. 00:36:45.260 --> 00:36:47.570 And they do this reversibly. 00:36:47.570 --> 00:36:50.480 OK, so the reversibility of this reaction 00:36:50.480 --> 00:36:54.380 is key for ATP PPI exchange to work. 00:36:57.430 --> 00:37:03.220 So if this occurs and they do this reversibly, 00:37:03.220 --> 00:37:09.310 therefore we can deduce formation of the aminoacyl AMP. 00:37:09.310 --> 00:37:14.320 If we add radio labeled PPI, the amino acid, and ATP 00:37:14.320 --> 00:37:18.670 to the enzyme and we see that radio labeled phosphorus 00:37:18.670 --> 00:37:24.010 from the radio labeled PPI incorporate into ATP. 00:37:24.010 --> 00:37:28.150 That's only going to happen if this chemistry is reversible. 00:37:28.150 --> 00:37:31.210 And bear in mind, we can detect very small quantities 00:37:31.210 --> 00:37:34.240 with radioactivity. 00:37:34.240 --> 00:37:36.520 So it's not that it has to be reversible 00:37:36.520 --> 00:37:38.350 to some large degree. 00:37:38.350 --> 00:37:42.550 We're relying on the detection of this radio label. 00:37:42.550 --> 00:37:45.700 So how does this work chemically? 00:37:45.700 --> 00:37:46.580 Let's take a look. 00:37:50.220 --> 00:37:54.930 OK, so imagine here we have our ATP. 00:37:54.930 --> 00:37:58.022 We have our amino acid. 00:37:58.022 --> 00:37:58.980 And we have our enzyme. 00:38:02.430 --> 00:38:04.275 And step one, we have binding. 00:38:09.300 --> 00:38:15.390 So there's some ATP binding site to the enzyme and some site 00:38:15.390 --> 00:38:16.800 for the amino acid to bind. 00:38:16.800 --> 00:38:19.470 And I'm leaving magnesium out of this depiction, 00:38:19.470 --> 00:38:22.950 but remember that magnesium and ATP come together. 00:38:22.950 --> 00:38:23.490 Now what? 00:38:23.490 --> 00:38:28.020 Step two, OK, we're going to have 00:38:28.020 --> 00:38:41.280 a chemical step where we have formation 00:38:41.280 --> 00:38:47.730 of the amino adenylate and PPI. 00:38:47.730 --> 00:38:50.295 And they currently are bound to the enzyme. 00:38:53.010 --> 00:38:53.975 We have step three. 00:38:59.130 --> 00:39:05.620 So imagine in this step our PPI is released. 00:39:12.600 --> 00:39:15.960 And this is another key aspect of this assay. 00:39:15.960 --> 00:39:17.520 So what does this mean? 00:39:17.520 --> 00:39:20.550 We now need to think about going backwards. 00:39:20.550 --> 00:39:25.620 If the PPI is released and we spike this reaction with radio 00:39:25.620 --> 00:39:28.980 labeled PPI and work our way backwards, 00:39:28.980 --> 00:39:32.660 will the radio label end up here in the ATP? 00:39:32.660 --> 00:39:33.660 OK. 00:39:33.660 --> 00:39:40.450 So this is going to be going backwards. 00:39:40.450 --> 00:39:47.260 We've left off with this enzyme with the amino adenylate bound. 00:39:47.260 --> 00:39:51.310 We have the PPI that was released. 00:39:51.310 --> 00:39:57.280 And then we spike this reaction with our radio labeled 00:39:57.280 --> 00:39:58.800 or hot PPI. 00:40:10.260 --> 00:40:12.000 So then what happens? 00:40:12.000 --> 00:40:15.480 Step four, working backwards. 00:40:21.610 --> 00:40:26.245 Imagine that some of the radio labeled PPI binds. 00:40:32.300 --> 00:40:34.340 Then what? 00:40:34.340 --> 00:40:35.930 Working backwards another step. 00:40:41.690 --> 00:40:45.075 32 P ATP and the amino acid. 00:40:51.910 --> 00:41:00.590 And then we have release here. 00:41:00.590 --> 00:41:04.180 OK, so then the question is, can you detect this? 00:41:12.250 --> 00:41:16.320 And so if you can detect some incorporation of this radio 00:41:16.320 --> 00:41:19.590 label into the ATP, that indicates 00:41:19.590 --> 00:41:23.163 that this enzyme worked through that type of intermediate. 00:41:26.544 --> 00:41:28.790 AUDIENCE: So are PPI not also sometimes 00:41:28.790 --> 00:41:34.340 [INAUDIBLE] and then if you had some competing hypothesis where 00:41:34.340 --> 00:41:39.240 it made ATP and ADP, then your PPI would maybe sometimes 00:41:39.240 --> 00:41:43.524 turn into just a single radio label 00:41:43.524 --> 00:41:46.350 phosphate that could then have the same reverse reactions 00:41:46.350 --> 00:41:47.823 as the [INAUDIBLE]? 00:41:47.823 --> 00:41:48.740 ELIZABETH NOLAN: Yeah. 00:41:48.740 --> 00:41:53.660 So whether you initially end up with PPI or PI 00:41:53.660 --> 00:41:57.770 is going to depend on how the ATP is hydrolyzed. 00:41:57.770 --> 00:42:01.250 And so you could imagine maybe there 00:42:01.250 --> 00:42:04.200 could be some background ATP hydrolysis 00:42:04.200 --> 00:42:07.700 that gives ADP and PI in this type of assay. 00:42:07.700 --> 00:42:11.492 That's something you always need to look out for. 00:42:11.492 --> 00:42:14.090 For the purpose of this, let's assume 00:42:14.090 --> 00:42:18.020 that we're not having some background problem in terms 00:42:18.020 --> 00:42:24.710 of the ATP source, and also that the enzyme is specific in terms 00:42:24.710 --> 00:42:28.130 of what it's doing to the ATP. 00:42:28.130 --> 00:42:31.670 But yeah, certainly background ATP hydrolysis 00:42:31.670 --> 00:42:34.110 can be a problem. 00:42:34.110 --> 00:42:39.560 So how will this be detected? 00:42:46.290 --> 00:42:51.550 And how will you know the radio label is associated with ATP 00:42:51.550 --> 00:42:53.175 and not something else in your mixture? 00:42:58.210 --> 00:42:59.160 AUDIENCE: [INAUDIBLE] 00:42:59.160 --> 00:43:00.700 ELIZABETH NOLAN: Pardon? 00:43:00.700 --> 00:43:01.627 AUDIENCE: [INAUDIBLE] 00:43:01.627 --> 00:43:02.460 ELIZABETH NOLAN: No. 00:43:02.460 --> 00:43:05.190 So we're going to look at the radioactivity. 00:43:05.190 --> 00:43:08.460 So this will come up more in recitation this week. 00:43:08.460 --> 00:43:11.850 But we need to be able to measure radioactivity by, say, 00:43:11.850 --> 00:43:14.640 scintillation counting here. 00:43:14.640 --> 00:43:16.710 But what's also needed is a separation 00:43:16.710 --> 00:43:19.800 because you need to know where that signal's coming from. 00:43:19.800 --> 00:43:22.710 You need to know it's coming from ATP 00:43:22.710 --> 00:43:26.070 and, say, not a background from however much of the PPI 00:43:26.070 --> 00:43:28.470 you introduced. 00:43:28.470 --> 00:43:31.590 Or if you have no idea what's going on with your chemistry, 00:43:31.590 --> 00:43:35.530 maybe the data are going to tell you it's not this mechanism. 00:43:35.530 --> 00:43:38.340 So you need to have a separation. 00:43:38.340 --> 00:43:41.910 So how might you separate ATP from all 00:43:41.910 --> 00:43:43.260 of these other components? 00:43:48.561 --> 00:43:50.020 AUDIENCE: Based on affinity column. 00:43:50.020 --> 00:43:51.900 ELIZABETH NOLAN: Some affinity column. 00:43:51.900 --> 00:43:54.150 So I like the column. 00:43:54.150 --> 00:43:57.810 But we're not going to have some sort of tag on the ATP. 00:43:57.810 --> 00:43:59.720 That might be a problem for that enzyme. 00:43:59.720 --> 00:44:01.830 But your notion is correct in the sense 00:44:01.830 --> 00:44:04.350 that we'll use some sort of chromatography 00:44:04.350 --> 00:44:06.230 in order to separate. 00:44:06.230 --> 00:44:08.700 OK, so maybe HPLC, how many of you 00:44:08.700 --> 00:44:12.613 have used an HPLC or at least know what one is? 00:44:12.613 --> 00:44:13.530 AUDIENCE: [INAUDIBLE]. 00:44:13.530 --> 00:44:14.488 ELIZABETH NOLAN: Right. 00:44:14.488 --> 00:44:17.640 So typically looking at UV vis. 00:44:17.640 --> 00:44:21.420 But you can imagine hooking up an HPLC to a detector that 00:44:21.420 --> 00:44:24.930 allows you to do scintillation counting and some sort 00:44:24.930 --> 00:44:29.760 of column that will allow you to look for ATP. 00:44:29.760 --> 00:44:35.660 Is all of the ATP going to be radioactive in this assay? 00:44:35.660 --> 00:44:37.560 No. 00:44:37.560 --> 00:44:40.740 So again, we can detect small quantities. 00:44:40.740 --> 00:44:43.260 And as long as there's a little bit of reversibility, 00:44:43.260 --> 00:44:46.380 we can see this here. 00:44:46.380 --> 00:44:50.190 OK, so what's critical in this assay 00:44:50.190 --> 00:44:53.160 is the reversibility of steps 3 and 4. 00:44:57.140 --> 00:45:00.170 What would happen in this assay if the PPI is not released? 00:45:06.783 --> 00:45:08.710 AUDIENCE: [INAUDIBLE]. 00:45:08.710 --> 00:45:09.828 ELIZABETH NOLAN: Right. 00:45:09.828 --> 00:45:11.620 Under the conditions, or if for some reason 00:45:11.620 --> 00:45:14.500 the PPI is not released, we're not 00:45:14.500 --> 00:45:17.350 going to see this exchange reaction. 00:45:17.350 --> 00:45:20.290 We're going to have a readout that doesn't give us this. 00:45:20.290 --> 00:45:21.700 Does that mean this didn't form? 00:45:24.620 --> 00:45:26.030 No. 00:45:26.030 --> 00:45:28.920 OK, so there's many caveats and details 00:45:28.920 --> 00:45:32.100 that you need to think through when thinking about a reaction 00:45:32.100 --> 00:45:36.220 and then the experiment is done to test this. 00:45:36.220 --> 00:45:40.620 So in the case of these aminoacyl-tRNA synthetases, 00:45:40.620 --> 00:45:44.970 these ATP PPI exchange assays work well. 00:45:44.970 --> 00:45:48.750 And these assays can be used to get steady state kinetic 00:45:48.750 --> 00:45:53.940 parameters, to get Kcat, Km, Kcat over Km, which 00:45:53.940 --> 00:45:58.890 is where this type of value comes from, in this case here. 00:46:05.490 --> 00:46:12.510 So back to these analyses up here, what they're telling us 00:46:12.510 --> 00:46:16.350 is that formation of this amino adenylate intermediate 00:46:16.350 --> 00:46:21.800 is about 60-fold faster than formation of the product. 00:46:21.800 --> 00:46:23.280 OK. 00:46:23.280 --> 00:46:25.110 And what we all want to recall when 00:46:25.110 --> 00:46:28.050 thinking about steady state experiments 00:46:28.050 --> 00:46:31.400 is that they're set up with a great excess of substrate 00:46:31.400 --> 00:46:33.960 and with the enzyme concentration. 00:46:33.960 --> 00:46:36.678 The reaction is zero order in respect to substrate. 00:46:36.678 --> 00:46:38.220 And you'll have some additional notes 00:46:38.220 --> 00:46:43.450 about that in your recitation materials this week for review. 00:46:43.450 --> 00:46:46.110 So something else biochemists like 00:46:46.110 --> 00:46:50.820 to do when looking at reactions and understanding reaction 00:46:50.820 --> 00:46:55.380 mechanisms is to look in the pre-steady state. 00:46:55.380 --> 00:46:57.870 And this came up briefly in lecture 1 as a method. 00:46:57.870 --> 00:46:59.370 And again, you'll hear more about it 00:46:59.370 --> 00:47:02.580 in recitation over the next two weeks. 00:47:02.580 --> 00:47:05.850 In these experiments, the goal is 00:47:05.850 --> 00:47:10.740 to look at the very first, early moments of a reaction. 00:47:10.740 --> 00:47:14.010 And they're set up quite differently. 00:47:14.010 --> 00:47:18.150 So limiting substrate is used. 00:47:18.150 --> 00:47:21.210 There's no turnover, so huge contrast 00:47:21.210 --> 00:47:24.570 to what we know about steady state experiments. 00:47:24.570 --> 00:47:27.870 And one of the goals is to look at the formation 00:47:27.870 --> 00:47:31.110 and consumption of intermediates here. 00:47:31.110 --> 00:47:36.080 So this type of chemistry often happens on a fast timescale. 00:47:36.080 --> 00:47:40.180 You can imagine millisecond timescale here, 00:47:40.180 --> 00:47:44.250 which means that we need a special apparatus that has fast 00:47:44.250 --> 00:47:46.920 mixing capabilities, because there's no way for one of us 00:47:46.920 --> 00:47:49.520 to do this on our own with our pipette. 00:47:52.350 --> 00:47:55.770 And so the type of experiment or apparatus 00:47:55.770 --> 00:47:58.020 used is called a stop flow. 00:47:58.020 --> 00:48:01.890 And I just show one depiction of a stop flow apparatus here. 00:48:01.890 --> 00:48:06.210 You'll get some other variations on this theme in the recitation 00:48:06.210 --> 00:48:07.530 notes. 00:48:07.530 --> 00:48:09.600 OK, but effectively what happens is 00:48:09.600 --> 00:48:14.120 that you have two drive syringes, a and b, 00:48:14.120 --> 00:48:17.700 and each of these syringes will contain certain components 00:48:17.700 --> 00:48:19.590 of your reaction. 00:48:19.590 --> 00:48:23.690 And this stop flow has a drive motor and a stop syringe. 00:48:23.690 --> 00:48:26.280 And it effectively allows you to rapidly mix 00:48:26.280 --> 00:48:31.860 the components of these syringes in a mixer, shown here. 00:48:31.860 --> 00:48:35.790 And then you either have some way to detect product-- 00:48:35.790 --> 00:48:38.430 so maybe if you can use optical absorption, 00:48:38.430 --> 00:48:43.470 you have a UV vis detector or a fluorescence detector. 00:48:43.470 --> 00:48:46.530 Or in other cases what you'll do is 00:48:46.530 --> 00:48:49.660 you'll punch the reaction at a certain time point. 00:48:49.660 --> 00:48:53.580 So you need a third syringe not shown here with a quencher. 00:48:53.580 --> 00:48:56.340 So you can imagine if you're working with an enzyme, 00:48:56.340 --> 00:49:00.120 maybe you quench by addition of acid or base, something 00:49:00.120 --> 00:49:03.390 that will denature and precipitate that enzyme. 00:49:03.390 --> 00:49:05.130 And then you can take that sample 00:49:05.130 --> 00:49:09.570 and analyze it in some way that fits in terms of what 00:49:09.570 --> 00:49:12.390 you need to detect there. 00:49:12.390 --> 00:49:17.190 So this type of methodology was used in order 00:49:17.190 --> 00:49:24.690 to monitor transfer of isoleucine to its tRNA. 00:49:24.690 --> 00:49:28.470 And so where we'll pick up in lecture on Wednesday 00:49:28.470 --> 00:49:33.210 is the design of that experiment in terms of what will we 00:49:33.210 --> 00:49:35.610 put in each syringe, and then what 00:49:35.610 --> 00:49:38.070 are the results of those experiments? 00:49:38.070 --> 00:49:42.900 And ultimately, what does that tell us about rates of transfer 00:49:42.900 --> 00:49:45.450 here? 00:49:45.450 --> 00:49:47.420 That's where we'll continue.